I am working with a 20 kDa protein which is kind of novel and not that much work has been done on this protein. By western blot, I some times get the band and most of the times can’t.
Last time I ran the same sample twice and side by side using DTT and 2ME. The lane that had been reduced by DTT showed a better result whereas the one with 2ME showed a kind of diffused band. I ran this western very short, like half an hour. I think this might be another reason that I got the band.
I know that both 2ME and DTT do not run with the protein and they are left behind in the top of the gel and if the protein is sensitive to oxidation, will be gone.
I heard there is other reducing agent which travels all the way with the running protein.
So I would be extremely appreciated if you could help:
1) Do you know any other reducing agent which can be run with the sample along the gel?
2) Why do you think when I run the gel longer the band gets disappeared?
Many thanks in advance